All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.
Considerable effort has been devoted to the search for therapies and diagnostics for cancers. The identification of cancer-specific molecules has remained the major stumbling block to the generation of suitable and effective therapies and diagnostics. One of the major reasons for this is the similarity between cancer cells and normal cells. It is possible that there are only very subtle variations in the proteins encoded during the formation of cancer cells. The search for these subtle variations is often driven by hope or inspiration, and entails considerable difficulty, expense and time.
Lung, colon and breast cancers are among the most common malignant tumours in humans. While methods of treatment have vastly improved in recent years, the effectiveness of treatment is critically dependent on early diagnosis. For example, while 70 to 80% of early detected, lymph node-negative breast cancer patients survive over 10 years after primary therapy, more than 60% die in this period if the tumour cells have already reached the lymph nodes before the commencement of treatment. The most efficient methods of early diagnosis for breast cancer are self-examination and mammography. No such method of early diagnosis is available for lung cancers, which can only be detected by radiography, which is usually carried out after symptoms become apparent, resulting in a much lower ten-year survival rate (approximately 13%). It is therefore generally accepted that improved, simple methods of early diagnosis would have a profound effect on the outcome of cancer treatments and mortality.
The most commonly used technique in clinical diagnosis in both veterinary and human medicine is the detection of antibodies or antigens in the serum of patients. The usefulness of serological tests for cancer diagnosis has however been disappointing, partly because cancer-specific or cancer-associated antigens have not been well defined and characterised. In addition, the use of serum as a source of specific antibodies has several inherent disadvantages, including:                1. The presence of large amounts of serum antibodies not related to the pathological agent, resulting in false positive results and background reactions;        2. The formation of antigen-antibody complexes in the serum which can disguise the presence of low titre antibodies/antigens;        3. The inability to detect antibodies produced locally at restricted mucosal sites, such as breast, colon and lung tissues, in the serum because of the massive dilution of these antibodies upon movement into the blood; and        4. The likelihood of persistence of specific antibodies in the serum, even in the absence of current disease makes it difficult to differentiate between previous and current disease.        
Prior art methods have primarily been directed to generation of monoclonal antibodies directed to cancer-specific antigens. For example, U.S. Pat. No. 5,093,261 by Hagiwara et al. discloses the generation of human hybridoma cell lines which produce monoclonal antibody specific for a primary liver cancer by fusion of lymph node lymphocytes from a patient with liver cancer with cells of a lymphoblastoid cell line. However, such methods are directed to making monoclonal antibodies against an unidentified antigen of a known cancer, rather than to identifying a cancer-specific antigen. In addition, monoclonal antibody production requires complex in vitro fusion and selection proceedings; the monoclonal antibodies thus produced do not reflect the total polyclonal immune response mounted against the cancer by the B cells in the draining lymph nodes. There is no disclosure or suggestion that the methods disclosed therein could be applied to preclinical diagnosis, staging of cancers, or detection of metastasis.
Therefore it was an objective of the inventors to develop methods for identifying antigenic molecules, or parts thereof, which are specific for individual cancers. Such molecules would be useful in the development of diagnostic or therapeutic agents for specific cancers.
We have previously shown that it is possible to identify protective antigens which are specific for pathogens such as parasites or bacteria of veterinary importance by culturing antibody-secreting cells from draining lymph nodes adjacent to a site of infection, isolating immunoglobulins from the culture medium, and using these immunoglobulins to identify pathogen-specific antigens. See for example U.S. Pat. No. 5,650,154; Meeusen and Brandon 1994a,b; and Walker et al, 1994, the entire disclosures of which are incorporated herein by this reference.
This method, which we have designated “ASC-probe technology”, utilises antibody-secreting cells as the source of the antibodies, and has been used extensively and successfully in the identification of new stage-specific and tissue-specific antigens and antibodies in endo- and ecto-parasite, bacterial and mycoplasmal infections (Meeusen and Brandon 1994a, b; Walker et al, 1994; Walker et al, 1996; Bowles et al, 1995; Bowles et al, 1996). This method also makes use of the versatility of antibodies to identify and detect antigens by commonly used methods, such as Western blotting and immunoprecipitation. The source of the antibodies is not serum but antibody-secreting cells (ASC). It has long been established that ASCs are induced in the local lymph nodes draining a disease-affected tissue, where antigen is deposited. From the lymph nodes, activated lymphocytes, including ASCS, migrate via the efferent lymphatic vessel through the bloodstream to the target tissue. The ASCs within the lymph nodes are short-lived, surviving for 4-6 days, and are only present as long as the antigenic stimulus is present within the tissue.
In addition, we have shown that the specific ASCs are restricted to the lymph nodes draining the affected organ or tissue, and that different lymph nodes within the same animal can react independently to different stages of infection with a pathogen, generally with different isotype and antigen recognition profiles (Meeusen and Brandon 1994b).
When cultured in vitro, the ASCs isolated from infected tissue or draining lymph nodes can be induced to secrete high levels of specific antibodies into the culture supernatant for several days. The antibody-containing supernatant, which we refer to as “ASC-probes”, is used directly to detect the presence and variety of antigens present at a particular time and tissue site and stage of infection. In addition to preparing ASC-probes from infected tissues and draining lymph nodes, we have also been able to isolate specific ASC-probes from circulating blood by making use of the narrow window of opportunity when ASCs are migrating from the lymph nodes to the tissue via the blood circulation. We have found that ASC-probes have distinct advantages over the use of serum antibodies for the discovery of novel pathogen-specific antigens useful for diagnosis and vaccine development.
In addition, the use of ASC-probes overcomes the major disadvantages of using serum antibodies mentioned above, in that:
1. ASCs are only generated after antigen stimulation, and the antibodies present in the ASC-probes are therefore predominantly specific for the disease agent, significantly reducing background and non-specific reactions;
2. As the cells used for ASC-probe preparation are washed free of serum before culture, there is no antigen present, and no antigen-antibody reaction which can reduce the sensitivity of the assay can occur;
3. ASCs locally produced in mucosal tissues can be isolated from tissue or from lymph nodes, or from peripheral blood during their migration to tissues;
4. As ASCs in lymph nodes are short-lived, they are only present as long as the antigenic stimulus is present in the tissue; the production of ASC-probes therefore reflects current infection or disease; and
5. ASC-probes, including those present in or secreted by peripheral blood lymphocytes, can provide a positive diagnosis, even at a stage when circulating antibody cannot be detected.
Since the development of this technique, there has been one publication reporting the use of peripheral blood ASCs for the detection of HIV infection in seronegative patients (Jehuda-Cohen et al. 1990).
While the use of ASC-probes has proven to be a major breakthrough in research relating to vaccines against infectious diseases(Meeusen and Maddox, 1999), this technique has not yet been applied to cancer research. The pathogenesis of cancers is very different from that of parasitic or bacterial infections, and cancer-specific protective responses are primarily T cell rather than B cell responses. Several studies have however reported the presence of activated B cells and plasma cells within draining lymph nodes and infiltrating lymphocytes of breast cancers (see for example Lynch and Houghton, 1993). However, there is no evidence as to whether this represents a specific response to a cancer-associated antigen, and the nature of these antigens has not been identified. Despite this, we believed that it was possible that cancer antigens might be specifically recognised by lymphocytes from adjacent lymphoid tissues. We therefore wished to determine whether the ASC-probe approach could also be successfully applied to cancer research, and whether ASC-probes could be used for the detection of novel tissue-specific and cancer-associated antigens, and for the early diagnosis of primary or recurrent cancer.